SUPPLEMENTARY MATERIALS

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1 SUPPLEMENTARY MATERIALS Bioactive compounds content and their biological properties of acetone extract of Cuscuta reflexa Roxb. grown on various host plants K. Tanruean a, P. Poolprasert a, J. Kumla b, N. Suwannarach b and S. Lumyong b* a Biotechnology Program, Faculty of Science and Technology, Pibulsongkram Rajabhat University, Phitsanulok, Thailand, b Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand, *Author for correspondence: saisamorn.l@cmu.ac.th (S. Lumyong), Tel: ext 144, Fax: , Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand, Abstract: This present study aimed to evaluate the total phenolic and flavonoid contents, and free phenolic compounds of acetone extract of Cuscuta reflexa grown on five different hosts including Coccinia grandis, Ficus racemosa, Samanea saman, Streblus asper and Zollingeria dongnaiensis and to explore the antioxidant activities, α-glucosidase and tyrosinase inhibitory properties of the extracts. The highest level of total phenolic and flavonoid contents were observed in the extract of Cu. reflexa that grown on St. asper (65.45 mg GAE/g extract) and Co. grandis (97.83 mg QE/g extract), respectively. According to HPLC results, vanillic acid, rutin and quercetin were found in all Cu. reflexa grown on diversified hosts. The extract of Cu. reflexa grown on Co. grandis possessed the greatest antioxidant activities (DPPH; µg/ml, FRAP; mg GAE/g extract), α-glucosidase inhibition accounted for percent and antityrosinase activity was at mg KAE/g sample. Keywords: Parasitic plant, antioxidant, antityrosinase, α-glucosidase inhibitory Experimental A. Chemicals All standards (purity >99.0%) for HPLC analysis were obtained from Sigma-Aldrich (St. Louis, MO, USA). Methanol, ethanol and acetonitrile were obtained from J.T.Baker (Radnor, PA,USA). Sodium carbonate, D-maltose, hydrochloric acid, aluminium chloride and folin ciocalteu s reagent were obtained from Fluka Chemie GmbH (Buchs, Switzerland). 2,2diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-tripyridyl-s-triazine (TPTZ), rat small intestinal

2 α-glucosidase, tyrosinase, PGO enzymes and 3,4-dihydroxy-1phenylalanine (L-DOPA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All the solvents and other chemicals were an analytical grade. B. Collection of plant material Cuscuta reflexa grown on Co. grandis, F. racemosa, Sa. saman, St. asper and Z. dongnaiensis were collected locally from Suthep District, Muang, Chiang Mai, Thailand. All plant samples were tentatively identified to species level by comparison with the reference materials at the herbarium of Queen Sirikit Botanic Garden, Chiang Mai, Thailand, and deposited as voucher specimens (PSRU-Conv ) in the collection of Faculty of Science and Technology, Pibulsongkram Rajabhat University (PSRU), Phitsanulok, Thailand. The plants were dried at 45 C, ground into powder and stored at room temperature for further extraction. C. Preparation of the extracts Twenty five grams of dried stem material of Cu. reflexa was individually extracted with 250 ml of acetone, and left overnight at room temperature. Afterwards, the extracts were put into ultrasonicator (Crest, USA) for 30 min, filtered through filter paper (Whatman no. 1) and evaporated at 40 C under a vacuum using a rotary evaporator until dry. Dry extracts were kept at room temperature in the dark prior to further study. D. Analysis of phenolic compounds by HPLC HPLC analysis of phenolic compounds was performed on a 1200 HPLC instrument (Agilent, Waldbronn, Germany) coupled to a quaternary pump and a UV detector by modifying the procedure of Fecka and Turek (2008) and Hossain (2010). The sample was separated on an Eclipe-C18 column (5.0 µm, mm). The mobile phase involved a solution of water with 0.1% formic acid in deionized water (A) and 100% acetonitrile (B). A gradient program was used according to the following profile: 0 10 min, 0 6 % B; min, increased to 19% B; min, increased to 100% B. The flow rate was 0.8 ml/min and the wavelength detection was recorded at 280 nm. The injection volume was 20 μl. Twenty one phenolic compounds including gallic acid, protocatechuic acid, catechin, vanillic acid, caffeic acid, syringic acid, chlorogenic acid, rutin, sinapic acid, ferulic acid, m-coumaric acid, hydroxycinnamic acid, ellagic acid, myrecetin, o-coumaric acid, rosmarinic acid, quercetin, luteolin, kaempferol, isorhamnetin and apigenin were analyzed at 280 nm.

3 E. Determination of total flavonoid contents The total flavonoid contents were determined following the method of Kaewnarin et al. (2014). The extract (0.5 ml) was mixed with 2 ml distilled water, followed by the addition 0.15 ml of 50 g/l NaNO 2. After 5 min, 0.15 ml of 100 g/l AlCl 3 was added. The reaction was mixed and incubated at room temperature for 15 min, and the absorbance was measured at 415 nm. Quercetin solution was used as a standard for the determination and the results were expressed as mg quercetin equivalent (QE)/g dry extract. The data were presented as the average of the triplicate analyses. F. Determination of total phenolic contents Total phenolic contents were estimated using the protocol of Thitilertdecha et al. (2008) with slight modifications. The procedure involved of combining 0.25 ml of sample (1 mg/ml) with 2.5 ml deionized water and 0.5 ml of folin-ciocalteu reagent. After 5 min, 0.5 ml of 20% (w/v) Na 2 CO 3 was added, and the solution was incubated for 1 hrs at room temperature. Absorbance was then measured at 760 nm. Gallic acid solution was used as a standard for the determination and the results were expressed as mg gallic acid equivalent (GAE)/g dry extract. The data were presented as the average of the triplicate analyses. G. Determination of α-glucosidase inhibitory activity α-glucosidase (AGH) solution was prepared from rat intestinal acetone powder by partial modification of the procedure reported by Oki et al. (1999). 100 mg of intestinal acetone powder was added to 3 ml of 0.9% NaCl solution, homogenized with the sonication and kept in an ice bath. After centrifugation at 6,000 rpm for 30 min at 4 C, the resulting supernatant was kept cold and directly subjected to inhibitory assay. The method of Adisakwattana et al. (2009) was used to determine AGH inhibitory assay. The assay was defined as the percent inhibition under the assay conditions, which was calculated according to the equation: Percent inhibition = [(A o A s )/ A o ] 100 Where A o is the absorbance of the control, and A s is the absorbance of the mixture containing the test compounds. The data were presented as the average of the triplicate analyses. H. Determination of tyrosinase inhibitory activity

4 The monophenolase inhibitory activity of the extract from the mushroom was determined using L-DOPA as a substrate by modifying the method of Chang et al. (2007). The tyrosinase enzyme was prepared as 1,000 units/ml and was used in 0.04 ml. Then, the proper volume of the diluted sample was added with adjusting of 50 mm phosphate buffer ph 6.8 into 1.76 ml. The mixture was pre-incubated at 37 C for 5 min prior to adding 0.2 ml of the L-DOPA substrate. The reaction was carried out at 37 C for exactly 10 min. The measurement of the dopachrom product was conducted at 475 nm. Percentage tyrosinase inhibitory activity was calculated by using the following equation. The result was expressed as kojic acid equivalent (KAE) per gram of dry weight (DW). Tyrosinase inhibitory activity (%) = [(A 0 A 1 )/ A 0 ] 100 Where, A 0 refers to the absorbance of the control solution, and A 1 represents the absorbance of the sample. I. Determination of antioxidant activities I.1 DPPH free radical scavenging assay The free radical scavenging ability was determined according to the method of Gülçin et al. (2003) with slight modifications. 2,2 -Diphenyl-1-picrylhydrazyl radical (DPPH ) solution in ethanol (0.1 mm, 1.5 ml) was mixed with 0.5 ml of different concentrations of each extract, and methanol was used as the control. The mixtures were well shaken and kept at room temperature for 30 min in the dark. The absorbance was measured at 517 nm. The percent of DPPH discoloration of the samples was calculated according to the formula: Percent inhibition = [(A o A s )/ A o ] 100 Where A o is the absorbance of the control, and A s is the absorbance of the mixture containing the test compound. The test sample concentrations providing 50% inhibition (IC 50 ) were calculated from the plot of inhibition percentage against extract concentration values. The radical scavenging ability was presented IC 50 values. The data were presented as the average of the triplicate analyses. I.2 Ferric reducing antioxidant power (FRAP) assay The FRAP assay was determined according to the protocol of Li et al. (2006) with some modifications. The FRAP reagent containing 10 mm of 2,4,6-tris(2-pyridyl)-s-triazine

5 (TPTZ) solution in 40 mm hydrochloric acid (20 ml), 20 mm ferric (III) chloride (20 ml) and acetate buffer (5 ml, 300 mm, ph 3.6) was prepared freshly prior to being used. Different concentrations of each extract (0.1 ml) was mixed with the FRAP reagent (1.5 ml) and 1.4 ml of acetate buffer (300 mm, ph 3.6) and were then incubated at an ambient temperature for 30 min. The absorbance was measured at 593 nm. Gallic acid was used as a standard and FRAP value was calculated as the gallic acid equivalent (mg GAE/g dry extract). The data were presented as the average of the triplicate analyses. J. Statistical analysis The results of all experiments were expressed as mean ± standard deviation. Analysis of variance was performed by ANOVA procedure and Duncan s multiple comparison test was used to determine any significant differences (P 0.05) identified between treatments. References Adisakwattana S, Chantarasinlapin P, Thammarat H, Yibchok-Anun S A series of cinnamic acid derivatives and their inhibitory activity on intestinal α-glucosidase. J Enzym Inhib Med Chem. 24: Chang TS, Ding HY, Tai SSK, Wu CY Mushroom tyrosinase inhibitory effects of isoflavones isolated soygerm koji fermented with Aspergillus oryzae BCRC Food Chem. 105: Fecka I, Turek S Determination of polyphenolic compounds in commercial herbal drugs and spices from Lamiaceae: thyme, wild thyme and sweet marjoram by chromatographic techniques. Food Chem. 108: Gülçın İ, Oktay M, Kıreçcı E, Küfrevıoǧlu Öİ Screening of antioxidant and antimicrobial activities of anise (Pimpinella anisum L.) seed extracts. Food Chem. 83: Hossain MB, Rai DK, Brunton NP, Martin-Diana AB, Barry-Ryan C Characterization of Phenolic Composition in Lamiaceae Spices by LC-ESI-MS/MS. J Agric Food Chem. 58: Kaewnarin K, Niamsup H, Shank L, Rakariyatham N Antioxidant and antidiabetic activities of some edible and medicinal plants. Chiang Mai J Sci. 41:

6 Li Y, Guo C, Yang J, Wei J, Xu J, Cheng S Evaluation of antioxidant properties of pomegranate peel extract in comparison with pomegranate pulp extract. Food Chem. 96: Oki T, Matsui T, Osajima Y Inhibitory effect of α-glucosidase inhibitors varies according to its origin. J Agric Food Chem. 47: Thitilertdecha N., Teerawutgulrag A, Rakariyatham N Antioxidant and antimicrobial activities of Nephelium lappacium L. extracts. LWT Food Sci Technol. 41: Table S1. Total phenolic and total flavonoid contents of the acetone extract of Cuscuta reflexa grown on five different hosts Host plant Total phenolic contents (mg GAE/ g extract) Total flavonoid contents (mg QE/ g extract) Coccinia grandis 49.49±2.65b 38.79±1.09a Ficus racemosa 43.33±1.39c 32.53± 1.26a Samanea saman 88.74± 1.41c 89.37±2.88b Streblus asper 65.45±3.17a 77.90±2.74b Zollingeria dongnaiensis 93.73± 1.85d 39.47± 3.41c * Average ± standard deviation from three replicates. The different letters in the same column are considered significantly different according to Duncan s multiple comparison test (P 0.05). Table S2. Phenolic compounds content in the extract of Cuscuta reflexa grown on five different hosts Compounds Retention times Content (mg/g extract) Co. grandis F. racemosa Sa. saman Gallic acid ±0.03a ND ND Vanillic acid ±0.07b 0.51±0.02c Rutin ±0.35b 2.50±0.01d Quercetin ±0.40a 2.10±0.07d 0.29±0.01 c 0.32±0.00 e 1.56±0.07 e St. asper 0.12±0.02 b 2.85±0.05 a 16.06±0.2 8a 13.77±0.0 9b Z. dongnaiensis ND 0.55±0.00c 2.70±0.07c 7.79±0.08c Values are expressed as means ± S.D. The different letters in the same row are considered significantly different according to Duncan s multiple comparison test (P 0.05).

7 Table S3. Biological activities of the acetone extract of Cuscuta reflexa grown on five different hosts Antioxidant activity α- Antityrosinase FRAP value Glucosidase activity Host plant DPPH assay (mg GAE/g inhibition (mg KAE/g sample) (IC 50 g/ml) extract) (%) Co. grandis ±3.14a 26.44±0.92a 84.36±1.21a 18.29±0.36a 80.57±0.73 F. racemosa ±5.55d 23.47±0.42b 17.98±0.68a b Sa. saman ±5.77c 23.81±0.41b 77.45±1.02c 6.18±0.93c St. asper ±4.97b 55.34±0.44b 71.59±2.41e 7.14±1.77bc Z ± ±5.35e 17.91±1.43c 8.30±0.58b dongnaiensis d Values are expressed as means ± S.D. The different letters in the same column are considered significantly different according to Duncan s multiple comparison test (P 0.05). Figure S1. HPLC chromatogram of standard phenolic compounds (GA, gallic acid; VA, vanillic acid; RU, rutin; QU, quercetin) identified in the acetone extract of Cuscuta reflexa grown on (A) Coccinia grandis, (B) Samanea saman, (C) Ficus racemosa, (D) Streblus asper and (E) Zollingeria dongnaiensis

8 Figure S2. HPLC chromatogram of internal standard phenolic compounds (blue line: GA, gallic acid; VA, vanillic acid; RU, rutin; QU, quercetin) in the acetone extract of Cuscuta reflexa grown on Samanea saman (red line)